The production of phosphitylated compounds via the reaction of hydroxyl-containing compounds with phosphine reagents is a transformation that has found utility in the synthesis of a wide range of useful compounds. For example, applicants have recognized that such a transformation is useful in the synthesis of 3′-O-phosphoramidites from 5′-O-protected nucleosides, as shown generally in Scheme 1:
wherein, for example, X is hydrogen, alkoxy, —O-tert-butyldimethyl silyl (OTBDMS), —O-methoxy methyl (OMOM), 2′-O-methoxyethyl (2′-O-MOE), and the like; R′ is DMT, dimethoxytrityl, oligonucleotides and analogs thereof, and the like; R″ is alkyl, such as methyl and the like, or alkoxy, such as 2-cyanoethyl and the like; R′″ is diisopropylamine and the like; and B is moiety derived from adenine, cytosine, guanine, thymine, or uracil.
Phosphoramidites of the type formed via Scheme 1 can be advantageously coupled to prepare oligonucleotides, see for example U.S. Pat. No. 4,725,677 and Mellor, Thomas, “Synthesis of analogues of oligonucloetides”, J. Chem Soc., Perkin Trans. 1, 1998, 747–757 (both of which are incorporated herein by reference), which have a rising importance in the field of therapeutic and diagnostic applications including, for example, antisense drugs (as described in Crooke, S. T. Handbook of Experimental Pharmacology: Antisense Research and Application; Springer-Verlag, Berlin, (1998), incorporated herein by reference). To supply the growing demand for these oligonucleotides, there is a desire to improve the synthesis of nucleosidic phosphoramidites on a commercial scale (Noe, Kaufhold, New Trends in Synthetic Medicinal Chemistry, Wiley-VCh Weinheim, 2000, 261, incorporated herein by reference).
However, applicants have come to appreciate that conventional methods for preparing phosphitylated compounds, such as 3′-O-phosphoramidites, from hydroxyl-containing compounds are disadvantageous for several reasons. One disadvantage associated with many conventional methods is the required use of costly and/or hazardous activating agents/compounds. For example, in Beaucage and Carruthers, Tetrahedron Lett. 1981, 22, 1859 (incorporated herein by reference), 1H-Tetrazole is recommended as the most versatile phosphitylation activator. However, such an activator/reagent is both expensive and hazardous. (See, for example, Stull, Fundamentals of Fire and Explosion, AlChE Monograph Series, No. 10, New York, 1977, Vol. 73, 22, incorporated herein by reference). Due to the explosive nature of the nitrogen-rich heterocycle, special safety precautions are required for the handling of such composition. A less hazardous compound, 4,5-Dicyanoimidazole, has been shown to be useful in the production of certain nucleosidic phosphoramidites. Unfortunately, this compound is very expensive, and, in fact, tends to be prohibitively expensive with regard to its use in industrial processes. Phosphitylation activators derived from unsubstituted pyridine are disclosed, for example, in Gryaznov, Letsinger, J. Am. Chem. Soc. 1991, 113, 5876; Gryaznov, Letsinger, Nucleic Acids Res. 1992, 20, 1879; Beier, Pfleiderer, Helvetica Chimica Acta, 1999, 82, 879; Sanghvi, et al., Organic Process Research and Development 2000, 4, 175; and U.S. Pat. No. 6,274,725, issued to Sanghvi et al., all of which are incorporated herein by reference. However, these salts tend to be toxic and highly water soluble. Accordingly, cost-intensive waste water treatment equipment must be installed in systems using such activators.
Another disadvantage associated with many conventional methods for preparing phosphitylated compounds is the use of dichloromethane as the preferred solvent. Because dichloromethane tends to be environmentally unfriendly, relatively costly waste treatment equipment is required for use in conjunction with methods involving dichloromethane as solvent.
One potential approach to avoid at least some of the aforementioned disadvantages is in situ preparation of nucleosidic phosphoramidites without an additional activation step, as described, for example, by Zhang et al., U.S. Pat. No. 6,340,749 B1, for immediate use of the resulting solution on the solid support synthesizer. Unfortunately, such methods tend to be relatively inefficient, and the phosphoramidite solutions obtained via such methods tend to be unstable and unsuitable for storage.
Accordingly, applicants have recognized the need for new methods of producing phosphitylated compounds which avoid the disadvantages associated with conventional methods.